US11326174B2 - Engineered yeast strains enabling anaerobic xylose fermentation decoupled from microbial growth - Google Patents
Engineered yeast strains enabling anaerobic xylose fermentation decoupled from microbial growth Download PDFInfo
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to the production of biofuel. More particularly, the present invention relates to rerouting Protein Kinase A (PKA) signaling to control sugar and hypoxia responses to promote anaerobic xylose fermentation in engineered yeast.
- PKA Protein Kinase A
- Bioethanol can be generated from lignocellulosic (LC) sugars derived from cellulosic biomass of renewable and sustainable plant feedstocks.
- LC lignocellulosic
- Energy of cellulosic biomass is primarily stored as the recalcitrant polysaccharide cellulose, which is difficult to hydrolyze because of the highly crystalline structure, and in hemicellulose, which presents challenges because of its structural diversity and complexity.
- Many microbes cannot natively ferment pentose sugars (e.g., xylose) from complex lignocellulosic biomass, which is composed of cellulose, hemicellulose and lignin fractions.
- Xylose is a prevalent sugar in both woody and herbaceous plants and a major component of hemicelluloses. Bioconversion of both xylose and glucose is required for the production of cellulosic biofuels. To further complicate matters, plant biomass must be chemically, mechanically, or thermally pretreated prior to enzymatic hydrolysis ex situ in order to produce fermentable glucose and xylose monomers. Such pretreatment processes generate a diverse array of degradation products derived from plant cell walls, such as hemicellulose and lignin-derived acetate and aromatic molecules, many of which inhibit cellular metabolism in S. cerevisiae and induce microbial stress during hydrolysate fermentation. See Taylor et al., Biotechnology J.
- the present invention is largely related to the inventors' research efforts to better understand xylose utilization for microbial engineering.
- the invention relates generally to methods and compositions for digesting lignocellulosic material and more particularly to methods that involve exposing the material to genetically engineered Saccharomyces cerevisiae ( S. cerevisiae ).
- the present invention provides, in a first aspect, a recombinant yeast genetically engineered to ferment xylose and exhibit a decreased level of BCY1 protein activity, wherein the recombinant yeast provides increased rate of anaerobic xylose fermentation in the yeast relative to a recombinant yeast having the same genetic background but not exhibiting a decreased level of BCY 1 protein activity.
- a degradable tag is operably linked to BCY1 to decrease levels of BCY1 protein activity.
- the recombinant yeast has been genetically engineered to lack BCY1 protein activity by a mutation in the recombinant yeast's BCY1 gene.
- the recombinant yeast exhibits reduced cell growth as compared to a recombinant yeast having the same genetic background but not exhibiting a decreased level of BCY1 protein activity.
- the recombinant yeast is of the genus Saccharomyces.
- the recombinant yeast is Saccharomyces cerevisiae.
- the yeast produces ethanol at an increased rate relative to a recombinant yeast not exhibiting decreased levels of BCY1 protein activity.
- the increased rate of ethanol production occurs under anaerobic conditions.
- the disclosure encompasses a yeast inoculum, comprising: (a) a recombinant yeast, as described above; and (b) a culture medium.
- the disclosure encompasses a method for producing ethanol by anaerobic fermentation of xylose in yeast, comprising: (a) culturing under ethanol-producing conditions a recombinant yeast, as described above, and (b) isolating ethanol produced by said recombinant yeast.
- the disclosure encompasses use of a recombinant yeast, as described above, in the anaerobic fermentation of xylose to produce biofuel.
- the disclosure encompasses a recombinant yeast, as described above, for use in the anaerobic fermentation of xylose to produce biofuel.
- FIG. 1 illustrates the response to anaerobiosis in xylose-grown cells.
- A) Log 2(fold change) in mRNA and protein for cells grown ⁇ O 2 , on glucose (black) or xylose (colored), with linear fit (R 2 ) listed.
- FIG. 2 illustrates Azf1 and Mga2 regulation of anaerobic xylose responses.
- A Average log 2(fold change) in mRNA abundance of denoted genes as listed in the key.
- B-D Distributions of log 2(fold change) in mRNA abundances for Hap4 (B), Msn2/Msn4 (C), and Mga2 (D) targets that are affected by AZF1 overexpression and show a corresponding change in Y128 versus controls.
- Asterisks indicate significant difference compared to azf1 ⁇ versus WT cells (paired T-test).
- E-F E-F
- OD 600 circles
- xylose concentration squares
- ethanol concentration triangles
- strain Y133 marker-rescued Y1278 lacking (mga2 ⁇ , orange plot on the left) or over over-expressing (“OE”, green plot on the right) MGA2, and Y133 wild type (“WT”) or empty-vector control (black) during anaerobic growth on xylose.
- WT Y133 wild type
- FIG. 3 illustrates an inferred network regulating phosphorylation changes during anaerobic xylose growth.
- A) Modules of peptides are shaped and colored according to class (Class A, diamond; Class B, square) and increase (yellow) or decrease (blue) of phosphorylation change across the strain panel, as described in the text. Each module is labeled with the phospho-motif sequence, with small case letter representing the phosphorylated site and “..” indicating non-specific residues. Implicated kinase regulators are shown as purple circles; proteins whose peptides belong to each module are shown as smaller circles, color-coded by protein function as listed in the key. Note that proteins with multiple phospho-sites can belong to multiple modules.
- B) Average (n 3) and standard deviation of the relative in vitro phosphorylation of a PKA substrate for lysates from cells that can (Y128, Y184 bcy1 ⁇ , Y184 Bcy1-AiD) or cannot (Y22-3, Y127) use xylose anaerobically. Orange bars represent phosphorylation in the presence of PKA inhibitor H-89.
- C) Average (n 3) and standard deviation of sugar utilization rates for Y133 tpk1 ⁇ tpk3 ⁇ tpk2 as or Y133 tpk1 ⁇ tpk3 ⁇ TPK2 during anaerobic growth, in the presence (green) or absence (black) of 1-NM-PP1.
- E) Average (n 3) and standard deviation of xylose utilization rates for strains in the presence (+) or absence ( ⁇ ) of SNF1. Asterisks indicate significant differences according to the key (paired T-tests).
- FIG. 4 illustrates mutation of BCY1 decoupling growth from anaerobic xylose metabolism.
- FIG. 5 illustrates an integrative model incorporating transcript, phospho-protein, and metabolite changes across the strain panel. Map of central carbon metabolism. Each step is annotated with boxes indicating mRNA difference (left) or phosphorylation difference (middle) in Y128 versus Y22-3, or phosphorylation difference (right) in Y184 bcy1 ⁇ versus Y184 grown anaerobically on xylose, according to the key. Gray indicates no significant change, white represents missing data, and multi-colored blue/yellow boxes indicate multiple phospho-sites with different changes.
- Metabolites measured previously are colored to indicate an increase (orange) or decrease (magenta) in abundance in Y128 versus Y22-3 grown anaerobically on xylose. Reactions predicted to be active (orange) or suppressed (magenta) in xylose fermenting strains based on mRNA, protein, and/or metabolite abundances are highlighted. Hexose transporters marked with a star have been implicated in xylose transport.
- FIG. 6 illustrates proteome and transcriptome response to anaerobic xylose growth across the strain panel.
- A) Log 2 (fold change) in abundance of Frd1 and Osm1 proteins across all strains and growth conditions in response to anoxia.
- B) Log 2(fold change) in abundance of ANB1 mRNA across all strains and growth conditions in response to anoxia.
- C) Log2(fold change) in mRNA abundance of the 128 genes with a progressive increase anaerobic xylose induction, in Y22-3, Y127, and Y128 growing in glucose ⁇ O 2 and xylose ⁇ O 2 .
- FIG. 7 illustrates deletion and over-expression of AZF1 influencing growth and fermentation under anaerobic xylose conditions.
- A-F OD 600 (circles), sugar concentration (squares), and ethanol concentration (triangles) for Y133 (marker-rescued Y128) azf1 ⁇ (red), Y133 AZF1 over-expression (“OE”, blue), and Y133 wild type (“WT”) or empty-vector control (black) for different sugars and growth conditions as indicated.
- FIG. 8 illustrates AZF1 over-expression increasing xylose fermentation in a second strain background with Y128 mutations.
- OD 600 (circles), xylose concentration (squares), and ethanol concentration (triangles) for CEN.PK113-5D with mutations required for xylose metabolism (HO ⁇ ::ScTAL1-Cpxy1A-SsXYL3-loxP-isu1 ⁇ hog1Agre3 ⁇ ira2 ⁇ 7, Table 3) harboring the AZF1 over-expression plasmid (purple) or empty vector control (black).
- FIG. 9 illustrates transcriptomic analysis of AZF1 deletion and over-expression during anaerobic xylose fermentation.
- FIG. 10 illustrates relative phosphorylation differences for known and inferred PKA targets across the strains growing anaerobically in xylose.
- Heat map represents relative abundance of phospho-peptides across the panel. Each row represents a phospho-peptide as measured in strains (columns) grown in xylose with (left) and without oxygen (right). Data represent average phospho-peptide abundance relative to the mean abundance across all six data points, such that yellow indicates phospho-peptide abundance above the mean and blue indicates phospho-peptide abundance below the mean, according to the key.
- 3A that harbor an RxxS phospho-motif and fall into different categories described in the main text, including Class A (progressive increase/decrease) and Class B (Y128-specific response).
- FIG. 11 illustrates that SNF1 is required for anaerobic xylose fermentation.
- A-C OD 600 (A), xylose concentration (B), and ethanol concentration (C) for Y133tpk1 ⁇ tpk3 ⁇ tpk2as (blue) or Y133tpk1 ⁇ tpk3 ⁇ TPK2 (black) in the presence of 10 ⁇ M 1-NM-PP1 (dashed line) or DMSO control (solid line). Timing of 1-NM-PP1 or DMSO addition is indicated by a red arrow.
- E OD 600 (circles), xylose concentration (squares), and ethanol concentration (triangles) for Y184 (Y22-3 gre3 ⁇ isu1 ⁇ )AZF1 over-expression (“OE”, purple) or Y184 empty-vector control (black).
- F OD 600 (circles), xylose concentration (squares), and ethanol concentration (triangles) for Y184 ira2 ⁇ AZF1 over-expression (“OE”, purple) or Y184 ira2 ⁇ empty-vector control (black).
- FIG. 12 illustrates SNF1 is required for anaerobic xylose and glucose fermentation.
- A-B OD 600 (circles), xylose concentration (squares), and ethanol concentration (triangles) for Y184 (Y22-3 gre3 ⁇ isu1 ⁇ ) ⁇ SNF1 (A) and Y184 bcy1 ⁇ SNF1 (B) grown in xylose ⁇ O 2 .
- SNF1+strains are plotted in black and snf1 ⁇ strains are plotted in orange.
- FIG. 13 illustrates that deletion of BCY1 influences anaerobic xylose fermentation.
- A-B OD 600 (circles), xylose concentration (squares), and ethanol concentration (triangles) for Y132 (marker-rescued Y127) ⁇ BCY1 (A) and Y184 ira2 ⁇ BCY1 (B) during growth in xylose ⁇ O 2 .
- BCY1+strains are in black and bcy1 ⁇ strains are in green.
- C) Average (n 3) and standard deviation of sugar utilization rates are shown for each strain ⁇ BCY1. Asterisks indicate significant differences (paired T-test) as indicated.
- FIG. 14 illustrates that inhibition of growth does not promote anaerobic xylose utilization.
- OD 600 circles
- xylose concentration squares
- Y128 xylose concentration
- green xylose concentration
- FIG. 15 illustrates that Bcy1-AiD is stable in both glucose+O 2 and xylose ⁇ O 2 .
- Anti-actin antibody was used as a loading control.
- Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
- a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. Promoters that allow the selective expression of a gene in most cell types are referred to as “inducible promoters”.
- a “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
- a host cell that has been transformed or transfected may be more specifically referred to as a “recombinant host cell”.
- Preferred host cells for use in methods of the invention include yeast cells, particularly yeast cells of the genus Saccharomyces , more preferably of the species Saccharomyces cerevisiae.
- a polypeptide “substantially identical” to a comparative polypeptide varies from the comparative polypeptide, but has at least 80%, preferably at least 85%, more preferably at least 90%, and yet more preferably at least 95% sequence identity at the amino acid level over the complete amino acid sequence, and, in addition, it possesses the ability to increase anaerobic xylose fermentation capabilities of a host yeast cell in which it has been engineered and over-expressed.
- substantially sequence homology refers to DNA or RNA sequences that have de minimus sequence variations from, and retain substantially the same biological functions as the corresponding sequences to which comparison is made.
- hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
- stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11.
- a preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4 ⁇ sodium chlorine/sodium citrate (SSC), at about 65-70° C. (or hybridization in 4 ⁇ SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1 ⁇ SSC, at about 65-70° C.
- a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in 1 ⁇ SSC, at about 65-70° C. (or hybridization in 4 ⁇ SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 0.3 ⁇ SSC, at about 65-70° C.
- a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in 4 ⁇ SSC, at about 50-60° C.
- SSPE (1 ⁇ SSPE is 0.15 M NaCl, 10 mM NaH 2 PO 4 , and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1 ⁇ SSPE is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete.
- additional reagents may be added to the hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS) chelating agents (e.g., EDTA), Ficoll, PVP and the like.
- blocking agents e.g., BSA or salmon or herring sperm carrier DNA
- detergents e.g., SDS
- EDTA chelating agents
- Ficoll e.g., Ficoll, PVP and the like.
- an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH 2 PO 4 , 7% SDS at about 65° C., followed by one or more washed at 0.02M NaH 2 PO 4 , 1% SDS at 65° C., see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995, (or alternatively 0.2 ⁇ SSC, 1% SDS).
- Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide(s)” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
- polynucleotide(s) also includes DNAs or RNAs as described above that contain one or more modified bases.
- DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotide(s)” as that term is intended herein.
- DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. “Polynucleotide(s)” also embraces short polynucleotides often referred to as oligonucleotide(s).
- isolated nucleic acid used in the specification and claims means a nucleic acid isolated from its natural environment or prepared using synthetic methods such as those known to one of ordinary skill in the art. Complete purification is not required in either case.
- the nucleic acids of the invention can be isolated and purified from normally associated material in conventional ways such that in the purified preparation the nucleic acid is the predominant species in the preparation. At the very least, the degree of purification is such that the extraneous material in the preparation does not interfere with use of the nucleic acid of the invention in the manner disclosed herein.
- the nucleic acid is preferably at least about 85% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
- an isolated nucleic acid has a structure that is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes.
- An isolated nucleic acid also includes, without limitation, (a) a nucleic acid having a sequence of a naturally occurring genomic or extrachromosomal nucleic acid molecule but which is not flanked by the coding sequences that flank the sequence in its natural position; (b) a nucleic acid incorporated into a vector or into a prokaryote or eukaryote genome such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene.
- PCR polymerase chain reaction
- nucleic acids present in mixtures of clones e.g., as those occurring in a DNA library such as a cDNA or genomic DNA library.
- An isolated nucleic acid can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded.
- a nucleic acid can be chemically or enzymatically modified and can include so-called non-standard bases such as inosine, as described in a preceding definition.
- operably linked means that the linkage (e.g., DNA segment) between the DNA segments so linked is such that the described effect of one of the linked segments on the other is capable of occurring.
- Linkage shall refer to physically adjoined segments and, more broadly, to segments which are spatially contained relative to each other such that the described effect is capable of occurring (e.g., DNA segments may be present on two separate plasmids but contained within a cell such that the described effect is nonetheless achieved). Effecting operable linkages for the various purposes stated herein is well within the skill of those of ordinary skill in the art, particularly with the teaching of the instant specification.
- gene product shall refer to the biochemical material, either RNA or protein, resulting from expression of a gene.
- heterologous is used for any combination of DNA sequences that is not normally found intimately associated in nature (e.g., a green fluorescent protein (GFP) reporter gene operably linked to a SV40 promoter).
- GFP green fluorescent protein
- a “heterologous gene” shall refer to a gene not naturally present in a host cell (e.g., a luciferase gene present in a retinoblastoma cell line).
- homolog refers to a gene related to a second gene by descent from a common ancestral DNA sequence.
- the term, homolog may apply to the relationship between genes separated by the event of speciation (i.e., orthologs) or to the relationship between genes separated by the event of genetic duplication (i.e., paralogs).
- orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes.
- Parents are genes related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.
- biofuel refers to a wide range of fuels which are in some way derived from biomass.
- the term covers solid biomass, liquid fuels and various biogases.
- bioethanol is an alcohol made by fermenting the sugar components of plant materials and it is produced largely from sugar and starch crops.
- Cellulosic biomass, such as trees and grasses, are also used as feedstocks for ethanol production and the present invention finds its primary application in this specific field.
- ethanol can be used as a fuel for vehicles in its pure form, but it is usually used as a gasoline additive to increase octane and improve vehicle emissions.
- yeasts are eukaryotic micro-organisms classified in the kingdom Fungi. Most reproduce asexually by budding, although a few undergo sexual reproduction by meiosis. Yeasts are unicellular, although some species with yeast forms may become multi-cellular through the formation of a string of connected budding cells known as pseudohyphae, or false hyphae, as seen in most molds. Yeasts do not form a single taxonomic or phylogenetic grouping. The term “yeast” is often taken as a synonym for Saccharomyces cerevisiae , but the phylogenetic diversity of yeasts is shown by their placement in separate phyla, principally the Ascomycota and the Basidiomycota. The budding yeasts (“true yeasts”) are classified in the order Saccharomycetales.
- nucleotides that occur in the various nucleotide sequences appearing herein have their usual single-letter designations (A, G, T, C or U) used routinely in the art.
- references to Greek letters may either be written out as alpha, beta, etc. or the corresponding Greek letter symbols (e.g., ⁇ , ⁇ , etc.) may sometimes be used.
- the inventors used comparative multi-omics and network analysis across the strain panel to understand the mechanism of improved anaerobic xylose flux.
- the inventors discovered that the parent strain growing anaerobically on xylose but not glucose fails to activate the hypoxic response, whereas evolved strains restore the response, showing that sugar metabolism and oxygen sensing are connected.
- the inventors found that rewiring cellular signaling by up-regulating Protein Kinase A (PKA) in conjunction with Snf1 activation coordinates a cascade of regulatory events that mediate sugar and hypoxic responses during anaerobic xylose growth.
- PKA Protein Kinase A
- deleting the PKA regulatory subunit decouples division and metabolism by halting growth but promoting rapid anaerobic xylose conversion. This provided an opportunity to distinguish proteome-wide phosphorylation events related to xylose-dependent growth versus fermentation.
- the inventors also found that simply fusing the regulatory subunit to a peptide tag combined the disparate benefits of wild-type and mutant strains, improving aerobic glucose growth as well as anaerobic xylose fermentation. Integrating transcriptomic, phosphoproteomic, and metabolomic data revealed a picture of the metabolic logic behind the improved flux of a non-native sugar.
- the present invention is a modified yeast strain lacking BCY1 protein activity and capable of anaerobic fermentation of xylose into ethanol without the need for cell growth.
- Multi-omic network analysis across strains The inventors used transcriptomic, proteomic, and phosphoproteomic profiling and bioinformatic analysis to study a series of yeast strains engineered and evolved for different levels of xylose fermentation.
- Strain Y22-3 is engineered with the minimal genes (xylose isomerase and xylulo-kinase) required for xylose utilization, but cannot use xylose anaerobically.
- Strain Y127 was evolved in the laboratory from Y22-3, and while it can respire xylose aerobically, it cannot ferment xylose anaerobically.
- Strain Y128 was evolved from Y127 and is capable of fermenting xylose to ethanol under anaerobic conditions. By comparing multi-omic profiles across these strains, the inventors implicated physiological responses that occur in Y128 but not the other strains to understand the bottlenecks in anaerobic xylose fermentation.
- PKA pathway signaling Phosphoproteomics and signaling network analysis implicated proteins in the PKA pathway as responsible for many changes in phosphorylation in Y128 fermenting xylose anaerobically.
- the inventors deleted BCY1, which serves as a regulatory protein turning PKA activity off, in a series of yeast strains known to be able to use xylose as an energy source. They found that in every strain tested, deleting BCY1 inhibited cell growth but enabled cells to consume xylose and produce ethanol anaerobically.
- deletion of BCY1 increased the amount of ethanol produced per yeast cell and per gram of sugar, thereby increasing theoretical yield of ethanol production, and also significantly increased the rate of xylose consumption.
- PKA is a hetero-tetramer made up of two catalytic subunits (encoded by any of three genes, TPK1, TPK2, TPK3) and two subunits of the regulatory protein BCY1.
- the secondary messenger cAMP is produced by adenylate cyclase and binds BCY 1 to inhibit its repressive regulation of the catalytic subunits, allowing TPK1, TPK2, and/or TPK3 to phosphorylate downstream targets.
- PKA signaling promotes glycolysis, cell growth, and ribosomal protein transcription and represses the stress response, gluconeogenesis, production of carbohydrate storage proteins glycogen and trehalose, and autophagy. While it was previously thought that cAMP binding by BCY 1 led to BCY 1 dissociation, recent work suggests PKA can remain active with BCY 1 still bound, suggesting a more complex regulatory interaction between the catalytic and regulatory subunits.
- PKA can be activated by at least two upstream branches, including the G-protein coupled receptors and RAS1/RAS2 signaling.
- IRA2 and its paralog IRA1 are the GTPase-activating proteins that negatively regulate GTP-activated RAS signaling; thus, deletion of IRA2 is expected to up-regulate RAS and, consequently, PKA signaling.
- SNF 1 which regulates metabolism of alternate carbon sources (and thus SNF 1 is usually inactive in the presence of glucose).
- SNF 1 phosphorylates myriad targets, including the glucose-responsive regulators MIG1 and RGT2 and other targets. Together, this results in derepression of gluconeogenic genes, respiratory genes, and genes encoding products allowing the cell to utilize alternative carbon sources in the absence of glucose.
- PKA and SNF 1 are thought to be active under different situations: PKA is active in the presence of ample nutrients and cellular energy, whereas SNF1 is active during energy limitation due to non-preferred carbon availability.
- the kinases are thought to have opposing effects on several shared regulators.
- the RGT1 transcriptional repressor controls hexokinase 2 (HXK2) transcription; RGT1 phosphorylation by SNF1 activates its repressive effects at the HXK2 promoter, whereas hyperphosphorylation by PKA inhibits RGT1 activity to de-repress this critical regulator of glycolytic flux.
- HXK2 hexokinase 2
- Lignocellulosic plant biomass is a renewable substrate for biofuel production, but many microbes cannot natively use the pentoses that comprise a large fraction of the sugars.
- Budding yeast Saccharomyces cerevisiae a key microbe in industrial biofuel production, is among the microbes that do not natively recognize xylose as a fermentable sugar, and even when engineered with conversion enzymes strains display low xylose utilization rates.
- Many studies have attempted to improve xylose metabolism, for example by optimizing xylose metabolism proteins, mutating or over-expressing xylose transporters, inducing genes in the pentose-phosphate pathway, or deleting pathways that siphon intermediates.
- strain Y22-3 was minimally engineered with xylose isomerase and other genes required for xylose metabolism but was unable to metabolize xylose.
- This strain was passaged aerobically on xylose-containing medium to produce the Y127 strain that respires xylose aerobically but cannot use xylose anaerobically.
- Y127 was thus further evolved without oxygen, generating strain Y128 that can ferment xylose to ethanol anaerobically with yields similar to other engineered strains (Table 1).
- Null mutations in iron-sulfur cluster scaffold ISU1 and the stress-activated HOG1 kinase enable xylose respiration in Y127, while additional loss of IRA2, an inhibitor of RAS/PKA signaling, and xylitol reductase GRE3 facilitate anaerobic xylose fermentation by Y1287.
- the IRA2 deletion is interesting, because it is expected to up-regulate RAS and Protein Kinase A (PKA) to promote growth: under optimal conditions, yeast maintain high PKA activity via increased cAMP that inactivates the PKA regulatory subunit Bcy1.
- mutations identified in Y128 promote xylose utilization in multiple strain backgrounds, and similar mutations were identified in an independent study, revealing that they have a generalizable impact on strains with the metabolic potential for xylose consumption. Furthermore, mutations in PKA regulators, including IRA2, frequently emerge in laboratory evolution studies that select for improved growth under various conditions. Yet the physiological impacts of these mutations that enable improved phenotypes, in particular anaerobic xylose fermentation, remain unclear.
- Multi-omic approaches have been used to characterize alternate sugar metabolism in engineered strains, but distinguishing causal cellular differences from secondary effects is generally a significant challenge. Comparative multi-omics across the strain panel have been used to distinguish transcript, protein, and phospho-protein differences that correlate with, and in several validated cases cause, improved xylose utilization. Integrating these results presents a systems-view of anaerobic xylose fermentation in yeast, which spans many individual responses that improve the phenotype. Our results support that augmenting cellular signaling to remodel many downstream effects that collectively improve the phenotype underlies the benefits in strain Y128. In the process of this work, the inventors present new insights into Snf1 and PKA signaling and the role of PKA mutations in laboratory evolutions.
- the inventors used comparative multi-omics across the strain panel to distinguish transcript, protein, and phospho-protein differences that correlate with improved xylose utilization capabilities, thereby implicating the mechanism of anaerobic xylose fermentation.
- the inventors first compared the transcriptome and proteome responses of parental strain Y22-3 and evolved strains Y127 and Y128 growing on glucose or xylose, with or without oxygen. Glucose-grown strains showed large changes in mRNA and their encoded proteins when shifted to anaerobosis, in all three of the strains ( FIG. 1A ).
- strains showed major differences when grown on xylose: Y22-3 shifted to anaerobic conditions showed large changes in mRNA but little change in the encoded proteins ( FIG. 1A ). Although this strain retained viability during the experiment, its inability to grow anaerobically could have caused a defect in protein production despite major mRNA induction upon the shift. In contrast, strain Y127 was unable to grow yet produced large mRNA changes and moderate changes in the corresponding proteins ( FIG. 1A ). This included several proteins (e.g. Pdr11 and Anb1) that are not expressed under aerobic conditions but were detected after the anaerobic shift, implicating nascent translation.
- Pdr11 and Anb1 proteins that are not expressed under aerobic conditions but were detected after the anaerobic shift, implicating nascent translation.
- strain Y127 and especially Y22-3 may have a defect in proteome remodeling during anaerobiosis despite large changes in mRNA, specifically when grown on xylose.
- Y22-3 and to some extent Y127 showed defective induction of ANB1, a canonical gene in the hypoxic response that is essential for anaerobic translation [40], whereas Y128 showed robust induction of ANB1 ( FIG. 6B ). While it remains unclear if defective ANB1 induction causes the defect in Y22-3 and Y127 protein accumulation, this observation led the inventors to discover that over 70% of genes classically involved in the hypoxic response (Table 2) were induced at the transcript level in all strains grown on glucose but largely uninduced in xylose-grown Y22-3 and induced progressively stronger in Y127 and Y128, respectively ( FIG. 1B ).
- the inventors identified 128 genes that were induced progressively stronger across the strain panel when shifted to anaerobic xylose conditions, with a pattern similar to the hypoxic response ( FIG. 6C ). These were enriched for genes involved in the hypoxic response, ergosterol biosynthesis, cysteine metabolism, and translation (p ⁇ 1 ⁇ 10-4, hypergeometric test).
- the inventors identified transcriptome effects of AZF1 deletion or over-expression.
- AZF1 over-expression in particular had broad effects on the anaerobic-xylose transcriptome, affecting 411 genes (FDR ⁇ 0.05) whose expression change also paralleled the difference in Y128 compared to Y22-3 grown anaerobically on xylose ( FIG. 9A ).
- Among the affected genes were several TFs and their targets.
- AZF1 also reduced expression of the gene encoding Mth1, which interacts with Rgt1 to repress hexose/xylose transporters ( FIG. 2A ); correspondingly, AZF1 over-expression induced several sugar transporters that can import xylose.
- MGA2 deletion decreased the rate of anaerobic xylose fermentation while MGA2 over-expression improved it ( FIG. 2E-2F ).
- the inventors were especially interested in the upstream regulatory network that mediates the downstream response, including activation of Azf1 and Mga2 targets.
- the inventors profiled the phosphoproteomes of Y22-3, Y127, and Y128 cultured on xylose with or without oxygen and then applied a novel network approach to infer regulation of strain-specific phosphorylation differences. Because many kinases recognize specific sequences around the phosphorylation site, the inventors identified ‘modules’ of phospho-peptides that are likely co-regulated and then implicated kinases and phosphatases that may control their phosphorylation change.
- the inventors grouped peptides based on their changes in phosphorylation when each strain was shifted from aerobic to anaerobic xylose conditions, identifying peptides with progressive phosphorylation increases or decreases across the strain panel (“Class A” increases or decreases) and peptides with responses uniquely higher or lower in Y128 (“Class B” increases or decreases).
- the inventors partitioned each group into ‘modules’ of peptides that harbor similar sequences around the phosphorylated site ('phospho-motifs'). Module peptides therefore share the same phosphorylation pattern and similar phospho-motifs, and thus are enriched for peptides that are likely co-regulated.
- module peptides are regulated by the same upstream regulator(s)
- the inventors searched a background network of protein interactions for proteins that physically interact with more module peptides than expected by chance (FDR ⁇ 0.05).
- the inventors focused on kinases whose phosphorylation preference matches the module phospho-motif, thereby implicating those kinases as direct regulators of module peptides.
- the resulting network implicated several regulators in the anaerobic xylose response ( FIG. 3A ).
- Peptides that showed highest phosphorylation levels in Y22-3 upon anaerobic xylose shift included ribosomal proteins and translation factors, whose modules were associated with PKA subunit Tpk2 and Cka1 of the CK2 kinase, which phosphorylates translation factors in other organisms to modulate translation.
- Other modules showed increased phosphorylation in Y128 shifted to anaerobic xylose conditions, including those connected to cyclin-dependent kinase Cdc28 that regulates carbon-metabolism enzymes and proteins required for division.
- this module also included hypoxia-responsive Mga2 at a site that matches the known PKA specificity.
- MGA2 genetically interacts with IRA2 in high-throughput datasets, supporting a link between PKA and MGA2 function.
- Mga2 targets are significantly up-regulated in Y128 that lacks functional IRA2 compared to nonfermenting strains ( FIG. 9B ). Together, these results suggest that signaling through PKA is modulated in Y128.
- Snf1 kinase which is activated by low cellular energy to induce alternative-carbon utilization genes.
- PKA and Snf1 are not normally active under the same conditions—the two regulators can produce antagonistic effects and even inhibit each other's activity.
- the inventors knocked out SNF1 from Y128 and measured xylose fermentation capabilities. Indeed, SNF1 is essential (but insufficient in the absence of other Y128 mutations) for anaerobic xylose utilization in Y128 ( FIG. 3D-3E, 13 ). Thus, PKA activity and SNF1 are required for the effect, validating the network predictions.
- IRA2 upregulates PKA as well as other downstream effects of RAS.
- the inventors deleted the PKA negative regulatory subunit BCY1 in strain Y184 (Y22-3 gre3 ⁇ isu1 ⁇ ) that can use xylose aerobically but not anaerobically.
- Y184 lacking BCY1 could not grow anaerobically on xylose, as seen for other non-preferred carbon sources—but surprisingly the cells rapidly fermented xylose despite growth arrest, at rates and ethanol yields surpassing other published xylose-converting strains ( FIG. 4A, 14 , Table 1).
- Xylose fermentation by the Y184 bcy1 ⁇ was associated with increased PKA activity, since lysate from Y184 bcy1 ⁇ cells grown anaerobically on xylose showed increased phosphorylation of a PKA target that was blocked by the H-89 PKA inhibitor ( FIG. 3B ).
- up-regulating PKA activity through BCY1 deletion enabled rapid xylose fermentation but in the absence of growth.
- the unique phenotype of the Y184 bcy1 ⁇ strain provided an opportunity to distinguish phosphorylation events related to growth versus metabolism. Phosphorylation patterns shared between Y184 and Y184 bcy1 ⁇ , neither of which can grow anaerobically on xylose, are therefore associated with growth arrest; in contrast, phosphorylation patterns common to Y184 bcy1 ⁇ and Y184 ira2 ⁇ , which share the ability to ferment xylose anaerobically but differ in growth capabilities, are implicated in xylose metabolism ( FIG. 4B -4D).
- FIG. 3A Several phosphorylation patterns implicated in Y128 ( FIG. 3A ) were not recapitulated in the Y184 bcy1 ⁇ strain, suggesting that they are not strictly required for anaerobic xylose fermentation. For example, unlike Y128, phosphorylation of known Cdc28 targets was reduced in Y184 bcy1 ⁇ compared to Y184 ira2 ⁇ , strongly suggesting that Cdc28-dependent phosphorylation in Y128 is linked to division and not xylose metabolism. Despite increased PKA signaling in the bcy1 ⁇ strain ( FIG. 3B ), several of the known and predicted PKA phosphorylation sites in Y128 showed reduced phosphorylation upon BCY1 deletion.
- Y184 bcy1 ⁇ showed decreased phosphorylation of serine 15 (S15) on the main hexokinase, HXK2, whose phosphorylation normally increases activity.
- S15 serine 15
- HXK2 main hexokinase
- the Y184 bcy1 ⁇ strain displayed several unique phosphorylation patterns not observed in the other strains. Remarkably, this included decreased phosphorylation on Hog1 activating site T174, a phenotype seen when Hog1 activity is reduced. This suggests that effects of BCY1 deletion mimic Hog1 inactivation that enhances xylose consumption, and raises the possibility that PKA activity can suppress Hog1 activation.
- BCY1 deletion enhances anaerobic xylose metabolism, it slows aerobic growth on glucose, which is a problem for industrial propagation of microbial cells.
- the inventors therefore generated a tagged version of Bcy1 in attempt to enable auxin-dependent degradation and made an important discovery: simply fusing a peptide to the carboxyl-terminus of Bcy1 (without enabling degradation) was enough to combine the benefits of BCY1+ and bcy1 ⁇ strains in aerobic and anaerobic conditions, respectively ( FIG. 4E-H ).
- the inventors' results provide new insight into the upstream regulatory network that enables anaerobic xylose fermentation and the downstream cellular responses that mediate it.
- S. cerevisiae engineered with the required metabolic enzymes remains unable to utilize xylose without further modification, indicating a bottleneck in regulation rather than metabolic potential. This bottleneck blocks metabolism but also prevents the hypoxic response, revealing for the first time a connection between sugar and oxygen sensing in yeast.
- Evolved strain Y128 activates PKA signaling while requiring Snf1 for anaerobic xylose usage, leading to a cascade of downstream effects that involve the sugar-responsive Azf1, oxygen-responsive Mga2, and downstream effectors that control respiration (Hap4), stress response (Msn2/Msn4), and sugar transport (Mthl) among others. Integrating transcriptomic, phosphoproteomic, and metabolomic data across the strain panel provides a glimpse of the downstream cellular response ( FIG.
- TAL1 transaldolase
- GDP1 glyceraldehyde-3-phosphate dehydrogenase
- ZWF1 glucose-6-phosphate dehydrogenase
- Snf1 activation induced expression of hexose transporters including some that transport xylose, altered hexokinase regulation, and Azf1 activation
- responses typically restricted to the presence of abundant glucose i.e. phosphorylation events associated with increased glycolytic flux, reduced expression of respiration and stress-responsive genes, and active PKA signaling.
- Snf1 is specifically required for anaerobic growth, on both xylose and glucose, in the context of Y128 mutations.
- Snf1 glucose-grown yeast exposed to hypoxia phase-separate glycolytic enzymes in a Snf1-dependent manner, in a process influenced by Ira2.
- Snf1 and PKA are not normally coactivated in yeast. The primary exception is during invasive growth, a foraging response in which starved cells invade a solid substrate. Invasive growth is driven by combined PKA and Snf1 activation, which is triggered by nitrogen and glucose limitation. This ecological response may explain the link between sugar and oxygen responses, since cells undergoing substrate invasion may prepare for impending hypoxia.
- Bcy1 is thought to direct PKA to specific sets of targets, much like AKAP proteins in mammalian cells.
- Integrating transcriptomic, phosphoproteomic, and metabolomic data across the strain panel also presents the downstream logic of anaerobic xylose fermentation ( FIG. 5 ).
- Transcriptomic and metabolomic data are easiest to interpret, and the combined effects in Y128 lead to induction of sugar transporters as well as genes and metabolites in the non-oxidative branch of the pentose phosphate pathway, increased abundance of xylolytic and glycolytic intermediates, reduced abundance of overflow metabolites, and sharp reduction in respiration components.
- Phosphorylation changes remain difficult to interpret in isolation, but the inventors propose that the integrative model shown in FIG. 5 can be used to predict the functions of corresponding phosphorylation changes and may have utility in future engineering strategies.
- the inventors' study demonstrates the power of integrative and comparative strategies to dissect cellular responses of an important industrial trait.
- Cells were grown in YP medium (10 g/L yeast extract, 20 g/L peptone) with glucose, xylose, or galactose added at 20 g/L final concentration, unless otherwise noted.
- Antibiotics were added where indicated at the following concentrations: 200 mg/L G418, 300 mg/L Hygromycin B, 100 mg/L ClonNat.
- For aerobic growth cultures were grown at 30° C. with vigorous shaking in flasks.
- media was incubated at 30° C. in a Coy anaerobic chamber (10% CO 2 , 10% H 2 , and 80% N 2 ) for ⁇ 16 hours before inoculation, and cultures were grown at 30° C.
- Saccharomyces cerevisiae strains used in this study are described in Table 3.
- the creation of Y22-3, Y127, and Y128 and their antibiotic marker-rescued counterparts with the KanMX gene was removed (Y36, Y132, and Y133, respectively) was described previously7.
- All strains express the minimal required genes for xylose metabolism, including xylose isomerase (xylA from Clostridium phytofermentans ), xylulose kinase (XYL3 from Scheffersomyces stipites), and transaldolase (TAL3 from S. cerevisiae ).
- xylose isomerase xylA from Clostridium phytofermentans
- XYL3 xylulose kinase
- TAL3 transaldolase
- Plasmid pST1933 (NBRP ID BYP8880) containing 3x Mini-AiD sequences, 5 ⁇ FLAG Tag and KanMX was modified to include the 329 bp of BYC1 3′ UTR between the 5 ⁇ FLAG tag and the KanMX marker gene.
- This construct (3 ⁇ Mini-AiD, 5 ⁇ FLAG tag, BCY1 3′ UTR, and KanMX) was amplified and inserted downstream and in-frame of BYC1 in Y184 (Y22-3 gre3 ⁇ isu1 ⁇ ) to form strain Y184 Bcy1-AiD. The integrated construct was verified by sequencing.
- Phenotypes introduced by BCY1 deletion were complemented by introducing BCY1 on a CEN plasmid: to generate the plasmid, BCY1 and 1000 bp upstream and 1000 bp downstream were amplified from Y128 and inserted into a NatMX-marked CEN plasmid via homologous recombination and sequence verified. This plasmid or the empty vector (pEMPTY) were transformed into appropriate strains. Phenotypes resulting from SNF1 deletion were complemented using the SNF1 MoBY 2.0 plasmid and compared to the empty vector control.
- Y133 tpk1 ⁇ tpk3 ⁇ tpk2 as was generated using CRISPR/Cas9-mediated genome editing.
- TPK1 and TPK3 were deleted in Y133 independently and verified by PCR.
- sgRNA sequence (GTGATGGATTATATCAGAAGG) that targeted the location within TPK2 to be replaced was cloned into the pXIPHOS vector using Not1 (GenBank accession MG897154), which contains the constitutive RNR2 promoter driving the Cas9 gene and NatMX resistance gene, using gapped plasmid repair using HiFi DNA Assembly Master Mix from NEB.
- tpk2as repair templates were generated by PCR of the whole ORF of tpk2as from a strain containing mutants of the TPK genes that are sensitive to the ATP-analogue inhibitor 1-NM-PP1 (TPK1 M164G, TPK2 M147G, TPK3 M165G). Purified repair templates were co-transformed at a 20-fold molar excess with the pXIPHOS-TPK2 sgRNA plasmid into Y133 tpk1 ⁇ tpk3 ⁇ strain.
- Colonies resistant to nourseothricin were restreaked onto YPD two times to remove the plasmid (and were verified to now be sensitive to nourseothricin) and tpk2as presence was verified by sequencing.
- Strains Y22-3 and Y127 were inoculated in rich xylose medium (YPX) at an OD 600 of ⁇ 0.5 and incubated anaerobically for the same amount of time as the other cultures. Y22-3 and Y127 retained over 95% viability as measured by CFU/mL after 17 hours of anaerobic incubation on xylose. Growth was halted by adding 30 mL of culture to ice cold 3.75 mL 5% phenol (pH ⁇ 5)/95% ethanol solution, cultures were spun for 3 min at 3000 rpm, the decanted pellet was flash frozen in liquid nitrogen and stored at ⁇ 80° C. until needed.
- YPX rich xylose medium
- RNA-seq library generation was performed using the Illumina TruSeq stranded total RNA kit (Illumina) using the sample preparation guide (revision C) with minor modifications, AMPure XP bead for PCR purification (Beckman Coulter, Indianapolis, Ind.), and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) as described in the Illumina kit. Libraries were standardized to 2 ⁇ M.
- Cluster generation was performed using standard Cluster kits (version 3) and the Illumina Cluster station. Single-end 100-bp reads were generated using standard SBS chemistry (version 3) on an Illumina HiSeq 2000 sequencer. All raw data were deposited in the NIH GEO database under project number GSE92908.
- Y133, Y133 azf1 ⁇ , Y133 with the AZF1 MoBY 2.0 plasmid, and Y133 carrying the MoBY 2.0 empty-vector control were grown in xylose ⁇ O 2 (+/ ⁇ G418 as needed), duplicate samples were collected on different days and RNA was isolated and DNA digested as described above. The inventors focused on genes affected in multiple strains for increased statistical power. rRNA depletion was performed using EpiCentre Ribo-Zero Magnetic Gold Kit (Yeast) RevA kit (Illumina) following manufacturer's protocols and cleaned using Qiagen RNease MinElute Cleanup kit (Qiagen, Hilden, Germany).
- RNA-seq library generation was performed using the EpiCentre Strand Specific ScriptSeq Kit (Illumina) as above except that Axygen AxyPrep Mag PCR Clean-up Kits for PCR purification (Axygen, Corning, N.Y.) were used and LM-PCR was performed using 12 cycles using EpiCentre ScriptSeq Index PCR Primers (Illumina) and Epicenter Failsafe PCR Enzyme Mix (Illumina). Single-end 100-bp reads were generated using standard SBS chemistry (version 4) on an Illumina HiSeq 2500 sequencer and the two FASTQ files for each sample were combined using the “cat” command.
- RNA-seq processing and analysis Reads for all RNA-seq experiments were processed with Trimmomatic version 0.351 and mapped to the Y22-3 genome using Bowtie 2 version 2.2.2 with default settings. HTSeq version 0.6.054 was used to calculate read counts for each gene using the Y22-3 annotation. Differential expression analysis was performed using edgeR version 3.6.8 using pairwise comparisons, taking Benjamini and Hochberg false discovery rate (FDR) ⁇ 0.05 as significant. Raw sequences were normalized using the reads per kilobase per million mapped reads (RPKM) method. Clustering analysis was performed using MClust version 4.457 and visualized using Java TreeView (http://jtreeview.sourceforge.net). Functional enrichment analysis was performed using the FunSpec database or a hypergeometric test using GO annotation terms (downloaded 2017-10-18). All examined targets of TFs were obtained from YeasTract using only those with DNA binding evidence.
- AZFI motif identification The inventors analyzed the log 2(fold change) in expression for each strain grown anaerobically in xylose compared to anaerobically in glucose. Genes with a progressive xylose-responsive induction across the strain panel were identified if the replicate-averaged log 2(fold-change) in Y127 was >1.5 fold higher than in Y22-3, and if the replicate-averaged log 2(fold-change) in Y128 was also ⁇ 1.5 fold higher than in Y127. 21 classical hypoxic genes, those known to be involved in the hypoxic response, were selected from the literature to measure the hypoxic response (Table 2) and for enrichment analysis to score the hypoxic response.
- the inventors selected 15 of these genes with no induction in Y22-3 grown anaerobically on xylose and performed motif analysis, by extracting 1000 bp upstream of these genes and submitting to MEME using the ‘any number of sequences’ model.
- the top motif matched the Azf1 binding site in TomTom. WebLogo was used to construct the final PWM logos for publication. Matches to this matrix were identified in 500 bp upstream regions in the Y22-3 genome using MAST with default settings. A total of 433 significant (E-value ⁇ 10) sites were identified in all intergenic regions in the genome.
- genes that showed an increase in expression when AZF 1 was over-expressed also showed an increase in expression in Y128 (relative to Y22-3), and vice versa.
- Functional enrichment analysis was performed using the FunSpec database or hypergeometric test of GO annotation terms (downloaded 2017-10-18) or compiled sets of TF targets.
- nLC-MS/MS For online nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS), reversed phase columns were packed-in house using 75 ⁇ m ID, 360 ⁇ m OD bare fused silica capillary.
- a nanoelectrospray tip was laser pulled (Sutter Instrument Company, Novato, Calif.) and packed with 1.7 ⁇ m diameter, 130 ⁇ pore size Ethylene Bridged Hybrid C18 particles (Waters) to a length of 30-35 cm.
- Buffer A consisted of 0.2% formic acid and 5% DMSO in water
- Buffer B consisted of 0.2% formic acid in acetonitrile. Two ⁇ g of peptides were loaded onto the column in 95% buffer A for 12 min at 300 min-1.
- Raw data was processed using MaxQuant version 1.4.1.268, and tandem mass spectra were searched with the Andromeda search algorithm.
- Oxidation of methionine was specified as a variable modification, while carbamidomethylation of cysteine was a set as a fixed modification.
- a precursor search tolerance of 20 ppm and a product mass tolerance of 0.35 Da were used for searches, and three missed cleavages were allowed for full trypsin specificity.
- Peptide spectral matches (PSMs) were made against a target-decoy custom database of the yeast strain was used, which was concatenated with a reversed sequence version of the forward database from McIlwain et al.
- FDR 1% false discovery rate
- Proteins were identified using at least one peptide (razor+unique), where razor peptide is defined as a non-unique peptide assigned to the protein group with the most other peptides (Occam's razor principle). Proteins were quantified and normalized using MaxLFQ with a label-free quantification (LFQ) minimum ratio count of 2. LFQ intensities were calculated using the match between runs feature, and MS/MS spectra were not required for LFQ comparisons. For quantitative comparisons, protein intensity values were log 2 transformed prior to further analysis.
- LFQ label-free quantification
- Phosphoproteomic analysis Phosphoproteomic experiments were multiplexed using tandem mass tags (TMT) isobaric labels to quantitatively compare the phosphoproteomes of Y22-3, Y127, and Y128 yeast strains. 6-plex experiments were performed to compare the three strains grown on xylose under aerobic and anaerobic conditions. Yeast phosphoproteomes were obtained from cell pellets from the same cultures used for the label free experiments described above using the same protein extraction, proteolytic digestion, and desalting conditions.
- TMT tandem mass tags
- a second phosphoproteomic experiment used TMT tags to compare the phosphoproteomic profiles of Y184, Y184 ira2 ⁇ , and Y184 bcy1 ⁇ during anaerobic growth on xylose in duplicate, using the same collection and methods outlined above.
- peptides from each condition were labeled with TMT 6-plex isobaric labels (Thermo Pierce) by re-suspending peptides in 200 ⁇ L of freshly made 200 mM triethylammonium biocarbonate (TEAB) and combining with 50 ⁇ L of the TMT labeling reagent resuspended in 100% acetonitrile.
- TEAB triethylammonium biocarbonate
- the samples were labeled for 4 hours, then ⁇ 5 ⁇ g of material from each TMT channel was combined into a test mix and analyzed by LC-MS/MS to evaluate labeling efficiency and obtain optimal ratios for sample recombination. Samples were quenched with 1.6 ⁇ L of 50% hydroxylamine, then combined in equal amounts by mass, and desalted.
- TMT-labeled peptides were then enriched for phospho-peptides using immobilized metal affinity chromatography (IMAC) with magnetic beads (Qiagen, Valencia, Calif.). After equilibration with water, the magnetic beads were incubated with 40 mM EDTA (pH 8.0) for 30 minutes while shaking. This process was repeated for a total of two incubations. Next, the beads were washed four times with water and incubated with 30 mM FeCl3 for 30 minutes while shaking, and this was also repeated for a total of two incubations. Beads were then washed four times with 80% acetonitrile/0.15% TFA.
- IMAC immobilized metal affinity chromatography
- TMT-labeled peptides were re-suspended in 80% acetonitrile/0.15% TFA and incubated with the magnetic beads for 45 minutes with shaking. Unbound peptides were collected for protein analysis. Bound peptides were washed three times with 80% acetonitrile/0.15% TFA and eluted with 50% acetonitrile, 0.7% NH4OH. Eluted peptides were immediately acidified with 4% formic acid, frozen, and lyophilized. Enriched phospho-peptides were re-suspended in 20 ⁇ L 0.2% FA for LC-MS/MS analysis.
- nLC-MS/MS Online nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS) was performed similarly as to the methods described above, including the same LC system and buffers, capillary reversed phase columns, gradient, and MS system and electrospray conditions. TMT phosphoproteomic experiments were also performed as single-shot (i.e., no fractionation) four-hour experiments.
- Variable modifications included oxidation of methionine; TMT 6-plex on tyrosine residues; phosphorylation of serine, threonine, and tyrosine residues; and neutral loss of phosphorylation on serine and threonine residues.
- a false discovery rate of 1% was used at the peptide and protein level.
- TMT quantification was performed and quantified peptides were grouped into proteins as described.
- Phospho-peptide localization was performed using phosphoRS73 integrated with COMPASS, using 75% as a localization probability cutoff to determine localized phospho-sites. Phospho-peptides with non-localized phospho-sites were discarded from further analysis.
- TMT reporter ion intensities were normalized for changes in protein level and log 2 transformed prior to further analysis.
- the PhosphoGRID database was used to identify phospho-sites of known function. All raw mass spectrometry files and associated information about identifications are available on Chorus under Project ID 999 and Experiment IDs 3016 and 3166.
- the inventors developed a novel network approach to infer kinases and phosphatases that mediate phosphoproteomic changes across the strain panel.
- the method predicts co-regulated groups of phospho-peptides, called modules, and then searches a background network of protein-protein interactions to identify ‘shared interactor’ proteins that physically interact with more module constituent proteins then expected by chance.
- the method consists of four steps: to identify potentially co-regulated peptides, the method 1) classifies phospho-peptides according to phosphorylation profiles across strains and then 2) within each class, partitions peptides into ‘modules’ of peptides that share the same motif around the phosphorylated site ('phospho-motif).
- the method identifies ‘shared interactor’ proteins that physically interact with more module constituents than expected by chance, and then 4) identifies the subset of shared interactors that are kinases and phosphatases, focusing on regulators whose known specificity matches the target module phospho-motif.
- Classifying Phospho-peptides Phospho-peptides. Phospho-peptides were partitioned into four classes based on the log 2(fold-change) in phosphorylation in each strain grown in xylose ⁇ O 2 versus xylose+O 2 . Class A contained phospho-peptides that show progressive increases or decreases in phosphorylation response (at least 1.5 fold difference in replicate-averaged log 2 expression changes, as described above) across Y22-3, Y127, and Y128.
- Identifying candidate module regulators The inventors focused on the subset of SIs that are kinases with known specificity and phosphatases whose interactions with the module were primarily directed toward module constituents or were undirected. For the kinases with known specificity, the inventors scored if the module phosphorylation motif matched the kinase motif as follows: Briefly, a position-weight matrix (PWM) was constructed for each module and compared to the PWM representing known kinase phosphorylation preferences from Mok et al.
- PWM position-weight matrix
- PWMs were generated from a peptide phosphorylation spot array assay where the normalized, background-corrected value is provided as a weight for each amino acid at each position, which was converted to a frequency value by calculating the total of all signal intensities for all amino acids at each position and then dividing by the total sum of the intensities. A pseudocount was used to prevent overfitting and to remove zeros that may occur in the Mok et al. PWMs.
- KLD Kullback-Leibler Divergence
- the inventors identified phospho-peptides with a reproducible log 2 expression difference of at least 1.5 ⁇ in both biological replicates in Y184 bcy1 ⁇ compared to Y184 (which mimics Y127) or in Y184 bcy1 ⁇ compared to Y184 ira2 ⁇ (which mimics Y128).
- Phospho-peptides were clustered using MClust version 4.457 and visualized using Java TreeView (http://jtreeview.sourceforge.net).
- Metabolomics analysis Metabolite data from Sato et al. was analyzed to compare changes in Y128 xylose ⁇ O 2 versus Y22-3 xylose ⁇ O 2 . A paired T-test was used to compare changes and those with a p-value ⁇ 0.05 were considered significant.
- PKA activity assay Measurement of PKA activity was performed on lysed cells using the PKA Kinase Activity Assay Kit from ABCAM. Cultures were grown anaerobically in xylose for three doublings (to OD ⁇ 0.5), at which 10 mL of cells were collected by centrifugation for 3 minutes at 3000 rpm, in preparation for lysis. Supernatant was removed under anaerobic conditions and the cells were resuspended in 1 mL of SB buffer (1 M sorbitol, 20 mM Tris HCl, pH 7.4) with 300 units of zymolyase (Amsbio) and 10 ⁇ L of protease inhibitor cocktail IV (Millipore).
- SB buffer (1 M sorbitol, 20 mM Tris HCl, pH 7.4
- Cells were incubated for 10 minutes at 30° C. anaerobically. Cells were collected by certification for 5 minutes at 350 ⁇ g and washed 1 ⁇ with SB buffer under anaerobic conditions. Cells were resuspended in 750 ⁇ L HLB buffer (10 mM Tris HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.3% (vol/vol) NP-40, 10% (vol/vol) glycerol) with 10 ⁇ L protease inhibitor cocktail IV and incubated on ice for 10 minutes, anaerobically. Cultures were subjected to ten rounds in a Dounce homogenizer anaerobically to promote lysis.
- HLB buffer 10 mM Tris HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.3% (vol/vol) NP-40, 10% (vol/vol) glycerol
- Lysis was verified using microscopy and total protein abundance was determined using a Bradford assay. 200 ⁇ L of cell lysate was removed and 50 ⁇ M H-89 was added as a PKA inhibitor and incubated for 10 minutes at 30° C. anaerobically.
- the PKA Kinase Activity Assay Kit was performed following manufacture's protocol, with the kinase reaction occurring under anaerobic conditions and the remaining steps (primary and secondary antibody incubation and washes) being performed aerobically. The reaction was detected using a TECAN Infinite 200 Pro with a wavelength of 450 nm. Positive (active PKA provided by ABCAM) and negative (no cells, blank) controls were used for each experimental reaction as verification of kit functionality. Relative PKA activity was calculated by subtracting the measured absorbance for each sample from the absorbance from the blank to remove background, followed by normalization to total protein abundance for each sample. Paired T-tests were used to determine significant differences among samples.
- Sugar consumption rate calculations Sugar consumption rates were calculated with a rate estimation tool as described previously using cell density (OD 600 ) and extracellular sugar concentrations measured by HPLC-RID. Log 2 data from each of three independent biological replicates were fit with linear models, and xylose consumption rate was calculated as g/L/OD/hour. Rates calculated for each replicate were plotted and compared with a paired T-test.
- BCY1-AID fermentations were designed to mimic high-cell titer industrial fermentations.
- Cells were grown in YP-6% glucose or YP-3% xylose to match sugar concentrations in hydrolysate.
- Strain Y184 Bcy1-AiD was grown aerobically in 6% glucose medium starting at an OD 600 0.1 or grown anaerobically in 3% xylose medium starting at OD 600 4.0. The tagged strain was compared to Y128, Y184 and Y184 bcy1 ⁇ . OD 600 and glucose, xylose, and ethanol were measured and rates were determined as described above.
- Bcy1-AiD stability was measured for each experiment using Western blot analysis as described previously.
- ⁇ -FLAG antibody (1:2500, Sigma) was used to detect Bcy1-AiD while ⁇ -Actin antibody (1:2500, Pierce) was used to detect actin as a loading control.
- the blot in FIG. 16 is representative of biological triplicates.
- the inventors compared phosphoproteomic data among three strains cultured anaerobically on xylose: Y184 (Y22-3 gre3 ⁇ isu1 ⁇ ) that can neither grow on nor metabolize xylose, Y184 ira2 ⁇ (Y22-3 gre3 ⁇ isu1 ⁇ ira2 ⁇ ) that both grows on and metabolizes xylose, and Y184 bcy1 ⁇ (Y22-3 gre3 ⁇ isu1 66 bcy1 ⁇ ) that does not grow on but metabolizes xylose.
- phospho-peptides showed a ⁇ 1.5 fold difference in abundance between Y184 bcy1 ⁇ and Y184 or Y184 ira2 ⁇ , in both biological replicates.
- These peptides were classified into three groups: Class I peptides are those with a difference in phosphorylation level in Y184 bcy1 ⁇ cultured anaerobically on xylose relative to both Y184 and Y184 ira2 ⁇ cultured under those conditions.
- Class II peptides are those with differences between the Y184 bcy1 ⁇ strain compared to Y184 ira2 ⁇ only—Class II peptides therefore represent those where the bcy1 ⁇ strain was more similar to Y184, neither of which grows anaerobically on xylose.
- Class III peptides are those whose phosphorylation was reproducibly different only in the bcy1 ⁇ strain compared to Y184, revealing that the bcy1 ⁇ strain behaved more like the Y184 ira2 ⁇ strain, which can also metabolize xylose ( FIG. 4B-4D ).
- the inventors examined each cluster using enrichment and network analysis, under the hypotheses that Class II phospho-peptides may relate to the growth defect of Y127 and Y184 bcy1 ⁇ whereas Class III phospho-peptides may be those associated with xylose metabolism, since both Y128 and Y184 bcy1 ⁇ can metabolize the sugar under these conditions.
- HXK2 is an interesting enzyme, because it acts both in glycolysis and as a regulator of nuclear transcription via the Mig1 repressor.
- Decreased phosphorylation at these sites may have broader effects here: deletion of HXK2 results in constitutive expression of Snf1 targets to enable growth on non-glucose fermentable carbon sources.
- the inventors' data raise the possibility that hexokinase activity is decreased to affect how the cell senses and/or responds to glucose availability.
- transketolase Tkl1
- Rnr2 the main deoxyribonucleotide-diphosphate reductase critical for nucleotide biosynthesis and thus growth.
- 28 phospho-sites in 24 proteins showed decreased in phosphorylation in the bcy1 ⁇ strain compared to Y184 ira2 ⁇ .
- Class III phosphorylation events were similar between Y184 bcy1 ⁇ and Y184 ira2 ⁇ strains, but distinct from Y184 that cannot metabolize xylose ( FIG. 4D ).
- the 51 phospho-sites (in 38 proteins) that displayed an increase in phosphorylation in the bcy1 ⁇ strain compared to Y184 were enriched for stress response proteins, including the Yak1 kinase that is antagonistic to PKA signaling and activated during times of stress3,4. Since PKA activity is known to suppress the stress response, this signature likely reflects PKA-dependent suppression of stress defense.
- Cdc25 the guanine-nucleotide exchange factor for RAS and a known PKA target.
- Increased phosphorylation on Cdc25 site S135 is thought to increase its activity, which would promote RAS-dependent signaling and PKA activity89.
- the increased phosphorylation of Cdc25 S135 is consistent with the notion that RAS/PKA activity is up-regulated by IRA2 or BCY1 deletion to promote increased xylose flux.
- Pkh1 involved endocytosis control
- Yck2 involved in morphogenesis, trafficking, and glucose response
- Akl1 endocytosis and cytoskeleton organization
- Ark1 regulation of actin cytoskeleton
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Abstract
Description
TABLE 1 |
Xylose consumption and ethanol production statistics comparing strains from this |
study to recently reported xylose fermentation strains in the literature. |
3 | 4 | 5 | |||||
Percent Xylose | Total Xylose | Total Ethanol | 6 | 7 | |||
Consumed | Consumed (g/L) | Produced (g/L) | Xylose | Xylose | |||
2 | (Total Time | (Total Time | (Total Time | Consumption | Consumption | 8 | |
1 | Culture | of Xylose | of Xylose | of Xylose | Rate | Rate | Ethanol Yield |
Strain | Conditions | Consumption) | Consumption) | Consumption) | (g/g/hr) | (g/OD/hr) | (g/g xylose) |
Y128 | Anaerobic | 98% (18 | 29.627 ± | 12.541 ± | 0.194 ± | 0.249 ± | 0.421 ± |
Batch, YP, | hours) | 0.095 (18 | 0.401 (18 | 0.020 | 0.017 | 0.014 | |
3% Xylose | hours) | hours) | |||||
Y184bcy1Δ | Anaerobic | 92% (18 | 27.503 ± | 12.178 ± | 0.219 ± | 0.263 ± | 0.446 ± |
Batch, YP, | hours) | 1.759 (18 | 0.167 (18 | 0.040 | 0.008 | 0.006 | |
3% Xylose | hours) | hours) | |||||
Y184Bcy1- | Anaerobic | 99% (18 | 29.709 ± | 12.378 ± | 0.260 ± | 0.334 ± | 0.445 ± |
AiD | Batch, YP, | hours) | 0.290 (18 | 0.290 (18 | 0.021 * | 0.019 * | 0.017 |
3% Xylose | hours) | hours) | |||||
H131-A3- | Anaerobic | — | — | — | 1.866 + | — | 0.41 |
ALcs | Batch, 2x | ||||||
YNB, 4% | |||||||
Xylose | |||||||
TMB3422 | Anaerobic | — | — | — | 0.580 + | — | 0.34 |
Batch, 2x | |||||||
YNB, 5% | |||||||
Xylose | |||||||
TMB3504 | Anaerobic | — | — | — | 0.760 + | — | 0.40 |
Batch, 2x | |||||||
YNB, 5% | |||||||
Xylose | |||||||
SXA-R2P-E | Anaerobic | — | — | — | 0.98 + | — | 0.45 |
Batch, | |||||||
Synthetic | |||||||
Medium, | |||||||
4% Xylose | |||||||
SR8 | Anaerobic | — | — | — | 0.87 ± | — | 0.31 ± |
Batch, | 0.02 + | 0.05 | |||||
YPX, 4% | |||||||
Xylose | |||||||
SR8- | Anaerobic | — | — | — | 0.31 ± | — | 0.351 ± |
fps1Δ | Batch, | 0.02 + | 0.001 | ||||
YPX, 4% | |||||||
Xylose | |||||||
RWB202- | Anaerobic | — | 20.627 ± | 8.306 ± | 0.34 + | — | 0.42 |
AFX | Batch, | 0.030 (42 | 0.060 (42 | ||||
Synthetic | hours) | hours) | |||||
Medium, | |||||||
2% Xylose | |||||||
RWB217 | Anaerobic | — | 20.102 ± | 8.684 ± | 1.06 + | — | 0.43 |
Batch, | 0.015 (42 | 0.060 (42 | |||||
Synthetic | hours) | hours) | |||||
Medium, | |||||||
2% Xylose | |||||||
LVY34.4 | Semi- | — | — | — | 1.32 + | — | 0.46 ± |
Anaerobic | 0.02 | ||||||
Batch, | |||||||
Synthetic | |||||||
Medium | |||||||
(YPX30), | |||||||
3% Xylose | |||||||
LVY41.5 | Semi- | — | — | — | 1.03 + | — | 0.45 ± |
Anaerobic | 0.02 | ||||||
Batch, | |||||||
Synthetic | |||||||
Medium | |||||||
(YPX30), | |||||||
3% Xylose | |||||||
BSPX021 | Anaerobic | — | 17.12 g/L | — | 0.217 ± | — | 0.39 ± |
Batch, | (72 hours) | 0.0.004 + | 0.00 | ||||
YNB, 2% | |||||||
Xylose | |||||||
36a(Bvu) | Oxygen | 71% (56 | — | 11.43 ± | 0.324 ± | — | 0.400 ± |
Limiting, | hours) | 0.02 (56 | 0.008 + | 0.001 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
36a(Xlq) | Oxygen | 66% (56 | — | 9.64 ± | 0.241 ± | — | 0.465 ± |
Limiting, | hours) | 0.06 (56 | 0.001 + | 0.001 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
36a(TAA) | Oxygen | 63% (56 | — | 10.15 ± | 0.333 ± | — | 0.400 ± |
Limiting, | hours) | 0.29 (56 | 0.000 + | 0.009 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
36a(HGB5) | Oxygen | 77% (56 | — | 12.27 ± | 0.261 ± | — | 0.400 ± |
Limiting, | hours) | 0.23 (56 | 0.002 + | 0.004 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
36a(YIT) | Oxygen | 64% (56 | — | 9.93 ± | 0.314 ± | — | 0.387 ± |
Limiting, | hours) | 0.16 (56 | 0.000 + | 0.007 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
39a(Bvu) | Oxygen | 92% (56 | — | 16.67 ± | 0.662 ± | — | 0.419 ± |
Limiting, | hours) | 0.08 (56 | 0.057 + | 0.003 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
39a(Xlq) | Oxygen | 78% (56 | — | 13.82 ± | 0.721 ± | — | 0.409 ± |
Limiting, | hours) | 0.98 (56 | 0.045 + | 0.014 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
39a(TAA) | Oxygen | 95% (56 | — | 16.35 ± | 0.532 ± | — | 0.401 ± |
Limiting, | hours) | 0.34 (56 | 0.011 + | 0.006 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
39a(HGB5) | Oxygen | 90% (56 | — | 15.63 ± | 0.704 ± | — | 0.402 ± |
Limiting, | hours) | 0.30 (56 | 0.005 + | 0.005 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
39a(YIT) | Oxygen | 91% (56 | — | 16.27 ± | 0.586 ± | — | 0.412 ± |
Limiting, | hours) | 0.01 (56 | 0.002 + | 0.005 | |||
Synthetic | hours) | ||||||
Medium, | |||||||
4% Xylose | |||||||
MA-B43 | Anaerobic | 78% (72 | — | — | — | — | 0.33 ± |
Batch, | hours) | 0.02 | |||||
Synthetic | |||||||
SC, 5% | |||||||
Xylose | |||||||
DLG-K1T2 | Anaerobic | 58% (72 | — | — | — | — | 0.37 ± |
Batch, | hours) | 0.00 | |||||
Synthetic | |||||||
SC, 5% | |||||||
Xylose | |||||||
DLK-K1T7 | Anaerobic | 85% (72 | — | — | — | — | 0.38 ± |
Batch, | hours) | 0.01 | |||||
Synthetic | |||||||
SC, 5% | |||||||
Xylose | |||||||
Entries marked with “--” indicate reported values were not measured with comparable units.
Column Descriptions:
|
|
Column | |
2 | Culture Conditions. Calculated from cultures started at an |
OD of 4.0 (FIG. 4). | |
|
Percent Xylose Consumed with Total Time of Xylose |
Consumption in | |
Column | |
4 | Total Xylose Consumed (g/L) with Total Time of Xylose |
Consumption in | |
Column | |
5 | Total Ethanol Produced (g/L) with Total Time of Xylose |
Consumption in Parentheses | |
Column 6 | Xylose Consumption Rate (g/g/hr) - Hours 6-16 for Y128, |
hours 0-18 for bcy1Δ and Bcy1- | |
Column | |
7 | Xylose Consumption Rate (g/OD/hr) - Hours 6-16 for Y128, |
hours 0-18 for bcy1Δ and Bcy1- | |
Column | |
8 | Ethanol Yield (g ethanol/g xylose) |
TABLE 2 |
Genes known to be involved in the hypoxic response used |
to examine the hypoxic response across the strain panel. |
1 | 2 | ||
| Gene | 3 | |
Name | Name | Description | |
YBR085W | AAC3 | Mitochondrial ADP/ATP translocator | |
YBR301W | DAN3 | Cell wall mannoprotein | |
YEL047C | FRD1 | Fumarate reductase required for anaerobic | |
growth | |||
YEL049W | PAU2 | Seripauperin multigene family | |
YER011W | TIR1 | Cell wall mannoprotein | |
YFL020C | PAU5 | Seripauperin multigene family | |
YHR210C | YHR210C | Putative aldose 1-epimerase protein | |
YIL011W | TIR3 | Cell wall mannoprotein | |
YIL013C | PDR11 | Sterol transporter | |
YJR047C | ANB1 | Anaerobic translation elongation factor eIF-5A | |
YJR051W | OSM1 | Fumarate reductase required for anaerobic | |
growth | |||
YJR150C | DAN1 | Cell wall mannoprotein | |
YJR151C | DAN4 | Cell wall mannoprotein | |
YKL197C | PEX1 | AAA-peroxin that recycles peroxisomal | |
targeting receptors under anaerobic conditions | |||
YLL025W | PAU17 | Cell wall mannoprotein | |
YLR037C | PAU23 | Cell wall mannoprotein | |
YML083C | YML083C | Protein of unknown function | |
YOL161C | PAU20 | Seripauperin multigene family | |
YOR009W | TIR4 | Cell wall mannoprotein | |
YOR010C | TIR2 | Putative cell wall mannoprotein | |
YOR011W | AUS1 | Sterol transporter | |
Column Descriptions:
|
Systematic name for each |
Column | |
2 | Standard name for each yeast gene, from the Saccharomyces |
| |
Column | |
3 | Gene functional description for each yeast gene, from the |
Saccharomyces Genome Database | |
TABLE 3 |
Strains used in this study. |
1 | |
Strain | 2 |
Name | Description |
Y22-3 | CRB Strain with xylose utilization genes (G418R) |
Y127 | Evolved Y22-3 for aerobic xylose utilization (G418R) |
Y128 | Evolved Y127 for anaerobic xylose utilization (G418R) |
Y36 | Y22-3 marker-rescued (MR) - lacking KanMX cassette |
Y132 | Y127 marker-rescued (MR) - lacking KanMX cassette |
Y133 | Y128 marker-rescued (MR) - lacking KanMX cassette |
Y133 | Y133 azf1Δ::KanMX (G418R) |
azf1Δ | |
Y133 | Y133 containing AZF1-MoBY 2.0 Plasmid (G418R) |
AZF1 | |
MoBY | |
Y133 | Y133 containing Empty Vector MoBY 2.0 Plasmid (G418R) |
MoBY | |
Control | |
CEN.PK1 | CEN.PK113-5D with HOΔ::ScTAL1-CpxylA-SsXYL3-loxP, isu1Δ::loxP, hog1Δ::kanMX, |
13-5D | gre3Δ::loxP, ira2Δ::loxP |
Xylose | |
Strain | |
CEN.PK1 | CEN.PK113-5D Xylose Strain containing AZF1-MoBY 2.0 Plasmid (G418R) |
13-5D | |
Xylose | |
Strain | |
AZF1 | |
MoBY | |
CEN.PK1 | CEN.PK113-5D Xylose Strain containing Empty Vector MoBY 2.0 Plasmid (G418R) |
13-5D | |
Xylose | |
Strain | |
MoBY | |
Control | |
Y184 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg (HygR) |
Y243 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg ira2Δ::MR (HygR) |
Y132 | Y132 bcy1Δ::KanMX (G418R) |
bcy1Δ | |
Y184 | Y22-3 gre3A::MR isu1Δ::loxP-Hyg bcy1Δ::KanMX (HygR, G418R) |
bcy1Δ | |
Y243 | Y22-3 gre3A::MR isu1Δ::loxP-Hyg ira2Δ::MR bcy1Δ::KanMX (HygR, G418R) |
bcy1Δ | |
Y133 | Y133 SNF1::Hyg (HygR) |
snflA | |
Y184 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg snf1Δ::NatMX (HygR, NatR) |
snf1Δ | |
Y184 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg bcy1Δ::KanMX snf1Δ::NatMX (HygR, G418R, NatR) |
bcy1Δsnf1Δ | |
Y243 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg ira2Δ::MR snf1Δ::NatMX (HygR, NatR) |
snf1Δ | |
Y243 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg ira2Δ::MR bcy1Δ::KanMX snf1Δ::NatMX (HygR, G418R, |
bcy1Δsnf1Δ | NatR) |
Y133 | Y133 snf1Δ::Hyg containing SNF1Δ-MoBY 2.0 Plasmid (HygR, G418R) |
snf1Δ | |
SNF1 | |
MoBY | |
Y133 | Y133 snf1Δ::Hyg containing Empty Vector MoBY 2.0 Plasmid (HygR, G418R) |
snf1Δ | |
MoBY | |
Control | |
Y133 | Y133 mga2Δ::KanMX (G418R) |
mga2Δ | |
Y133 | Y133 containing MGA2-MoBY 2.0 Plasmid (G418R) |
MGA2 | |
MoBY | |
Y184 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg bcy1Δ::KanMX containing pBCY1 CEN Plasmid (HygR, |
bcy1Δ | G418R, NatR) |
pBCY1 | |
Y184 | Y22-3 gre3Δ::MR isu1Δ::loxP-Hyg bcy1Δ::KanMX containing empty vector CEN Plasmid |
bcy1Δ | (HygR, G418R, NatR) |
Empty | |
Control | |
Y132 | Y132 BCY1-3′ AiD tag (3x Mini-Auxin Induced Degron Sequence-5x FLAG-BCY1-3′ UTR- |
BCY1-3′ | KanMX) (G418R) |
AiD | |
Y184 | Y22-3 gre3Δisu1Δ BCY1-3′ AiD tag (3x Mini-Auxin Induced Degron Sequence-5x FLAG- |
BCY1-3′ | BCY1-3′ UTR-KanMX) (G418R) |
AiD | |
Y243 | Y36 gre3Δisu1Δira2Δ BCY1-3′ AiD tag (3x Mini-Auxin Induced Degron Sequence-5x |
BCY1-3′ | FLAG-BCY1-3′ UTR-KanMX) (G418R) |
AiD | |
Column Descriptions:
|
| |
Column | ||
2 | Description of Strain Used | |
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